Hypoglycosylated recombinant glucose oxidases

ABSTRACT

Hypoglycosylated recombinant glucose oxidase with a molecular weight of ca. 68-80 kDa, a specific activity of ca. 200 U/mg unit of weight, a carbohydrate portion of ca. 12% which is obtainable by expression of a recombinant DNA containing the GOD gene in the N-glycosylation-defective yeast mutants DSM 7042, DSM 7338, DSM 7160 or DSM 7340 or allelic mutant strains, fermentation and isolation of the enzyme from the culture supernatant or the cells.

The invention concerns a hypoglycosylated recombinant glucose oxidase(GOD EC 1.1.3.4) as well as the production thereof and its use indiagnostic tests.

There are three ways in which a protein can be providedposttranslationally with carbohydrates. A distinction is made between:

N-glycosylation

N-glycosidic linking of the carbohydrate chain to Asn

O-glycosylation

O-glycosidic linking of the carbohydrate chain to Thr or Ser

glycosyl-phosphatidyl-inositol anchor (GPI)

component of some membrane proteins,

the GPI anchor serves to embed them in the phospholipid membrane.

The glycosylation of proteins is described for example in:

Kukuruzinska, M. A. et al., Ann. Rev. Biochem. 56 (1987) 915-944;

Paulson, C. P., TIBS 14 (1989) 272-276;

Warren, C. E., BFE 7 (1990) 392-395;

Ballou, C. E., In: Strathern, J. N., et al., The Molecular Biology ofthe Yeast Saccharomyces, Cold Spring Harbor Laboratory, New York, pp.355-360 (1982).

Kornfeld, R.; Kornfeld, S., Ann. Rev. Biochem 54 (1985) 631-664;

Tanner, W.; Lehle, L., Biochim. Biophys. Acta 906 (1987) 81-99;

Innis, M. A., In: Barr, P. J. et al., Yeast genetic engineering,Butterworths, Stoneham, Mass., pp. 233-246 (1989).

The O-glycosidic carbohydrate structures of yeast proteins consist of anunbranched mannose chain of 1-5 mannose residues. The O-glycosylationbegins in the ER (transfer of the first mannose residue) and iscompleted in the Golgi apparatus.

N-glycosylation takes place in two steps. A core unit ofN-acetylglucosamine, mannose and glucose is built on a lipid carrierintermediate and this is transferred in the ER onto Asn residues ofglycoproteins. After the protein-bound core unit has been processed(cleavage of the glucose residues and a specific mannose residue in theER), the sugar structure is elongated in the Golgi apparatus ("outerchain" glycosylation). The structure of the outer chain glycosylation isorganism-specific.

Glucose oxidase (GOD EC 1.1.3.4) from Aspergillus niger is a naturallysecreted N-glycosylated homodimer with a molecular weight of ca. 80 kDaper subunit (SU), 1 FAD as a cofactor/SU and one disulfide bridge/SU.GOD from A. niger has a relatively uniform carbohydrate structure (coreglycosylation).

The technical production of glucose oxidase from Aspergillus niger is,however, difficult. GOD in Aspergillus niger is evidently transportedinto the peroxisomes (Dijken, J. P. van and Veenhuis, M., Eur. J. Appl.Microbiol. 9 (1980) 275-283) which impedes the processing. However,under certain conditions the enzyme can also be secreted into the medium(Mischak, H. et al., Appl. Microbiol. Biotech. 21 (1985) 27-31). Theyield of GOD is only low in this case. For these reasons many attemptshave already been made to recombinantly produce the glucose oxidase fromAspergillus niger in Saccharomyces cerevisiae. The recombinant GOD fromSaccharomyces cerevisiae is more thermostable and pH-stable than thenative enzyme from Aspergillus (De Baetselier A. et al., Biotechnology 9(1991) 559-561). Although high yields of enzyme were obtained in therecombinant production, it turned out, however, that the recombinantenzyme is heterogeneous with regard to the portion of carbohydrate andto the molecular weight of 80 to 140 kDa/SU (SU=subunit) due to anon-uniform "outer chain glycosylation" of up to 150 mannose residues.The recombinant enzyme is hyperglycosylated (carbohydrate portion ca.70% instead of 16% as in the native enzyme) (De Baetselier et al.,Biotechnology 9 (1991) 559-561) and therefore has a substantially highermolecular weight (ca. 80-140 kDa/SU) than the native enzyme. This isdisadvantageous for the application of GOD especially in diagnostictests e.g. the hyperglycosylated recombinant enzyme has a lower specificactivity in units per unit of weight (ca. 65 U/mg compared to 178 U/mgfor the native enzyme from Aspergillus).

Kriechbaum, M. et al., FEBS Lett. 255 (1989) 63-66;

Frederick K. R. et al., J. Biol. Chem. 265 (1990) 3793-3802;

De Baetselier, A. et al., Biotechnology 9 (1991) 559-561;

Whittington, H. et al., Curr. Genet. 18 (1990) 531-536;

Rosenberg, S., WO 89/12675;

The sequence of the glucose oxidase from Aspergillus niger is alsodescribed in these publications.

The object of the present invention was to avoid these disadvantages andto provide a recombinant glucose oxidase the N-glycosylation of which isas uniform and low as possible (e.g. with a complete or partial defectin the outer chain glycosylation) and with a high specific activity.

This object is achieved by a hypoglycosylated recombinant glucoseoxidase with a molecular weight of 68-80 kDa, a specific activity of ca.200 U/mg unit of weight, a carbohydrate portion of ca. 12% which isobtainable by expression of a recombinant DNA containing the GOD genefrom Aspergillus in the N-glycosylation-defective yeast mutants DSM7042, DSM 7160, DSM 7338 or DSM 7340 or allelic mutant strains thereof,fermentation and isolation of the enzyme from the culture supernatant orthe cells.

It was surprisingly found that GOD isolated fromN-glycosylation-defective yeast mutants (e.g. ngd29 mutants) is muchmore stable than the enzyme from Aspergillus niger with a comparableportion of carbohydrate, molecular weight (68-80 kDa, preferably 68-75kDa) and specific activity.

The yeast strains which are suitable for producing the GOD according tothe invention and the production thereof are described in the GermanPatent Application P 42 26 094.9 with the same priority, the contents ofwhich are a subject matter of the disclosure of the present invention.

Such yeast strains are obtainable by [³ H]-mannose suicide selection,introduction of one or several selectable markers (auxotrophyrequirements and/or resistances) and selection of those strains which,after transformation with the plasmid YEpL/GOD, secrete more than 10mg/l GOD into the medium after culture under standard conditions and areallelic to the Saccharomyces cerevisiae mutants ngd29 (DSM 7042, DSM7338) or ngd62 (DSM 7160, DSM 7340).

The technique of [³ H]-mannose suicide selection essentially comprises:

mutagenesis (e.g. starting from the wild-type strain X2180-1A, ATCC26786)

incubation with [³ H]-mannose

accumulation of hyperglycosylation-defective mutants by storing thecells at -80° C. until the survival rate of the cells falls to 2-3powers of ten (2-4 months)

selection for mutants with reduced N-glycosylation on the basis ofhomologously expressed invertase

analysis by activity staining and/or

immunoprecipitation of secreted invertase and determination of themolecular weight (glycosylation) by SDS-PAGE

The auxotrophy markers are introduced by crossing the yeast strains toform diploids (isolation of the zygotes by micromanipulation) and ifnecessary subsequent sporulation to form haploids (tetrad analysis). Thengd phenotype was determined by activity staining of external invertaseby means of native PAGE gels using sucrose and2,3,4-trinitrophenyltetrazolium chloride as the substrate/glucosereagent.

Determination of an adequate GOD production can be carried out bydetermining the activity of the GOD secreted into the medium afterculture under standard conditions. For this the strain to be tested (GODtransformant), is incubated for 3-4 days while shaking preferably aftera selective preculture in complete medium. Yeast extract, Bactopeptone,fructose and maltose are preferably added at neutral pH to the completemedium.

The determination of glucose oxidase is carried out for exampleaccording to the method described in the examples under "generalmethods".

Mutants according to the invention (allelic mutants) can be determinedby a test in which the mutants to be tested are analysed whether theyhave a mutation in the same genes as the yeast strains DSM 7042/7338(ngd29) and DSM 7160/7340 (ngd62).

For this the strain to be tested is crossed in each case with the yeaststrains DSM 7042/7338 and DSM 7160/7340 and the diploid strains obtainedin this process are analysed.

The mutation (strain) to be tested is allelic to the ngd mutantsaccording to the invention (DSM 7042/7338, ngd29) and/or (DSM 7160/7340,ngd62) if the mutations do not compensate in diploid cells.

The mutation (strain) to be tested is not allelic to the ngd mutantsaccording to the invention (DSM 7042/7338, ngd29) and/or (DSM 7160/7340,ngd62) if the mutations complement themselves in the diploid cells and awild-type phenotype results with regard to N-glycosylation.

Yeast strains which are preferably used according to the invention arethe strains DSM 7042, DSM 7160, DSM 7338 and DSM 7340. The strains DSM7338 and DSM 7340 are particularly preferred.

The thermostability was determined on the basis of DSC spectra("differential scanning calorimetry"). According to this the Tm value isat least 73° C. compared to 68.5° C. for the GOD from Aspergillus niger.

After a temperature stress at 55° C. for 2 hours the residual activityof GOD is still at least 30%.

A preferred embodiment of the GOD according to the invention isC-terminal active GOD fusion proteins which contain a plurality ofadditional histidines or cysteines. Such derivatives are described forexample in EP-B 0 184 355 and can be purified in a simple manner.

The invention in addition concerns a process for the production ofrecombinant glucose oxidase from Aspergillus niger in Saccharomycescerevisiae with a uniform carbohydrate structure characterized in thatSaccharomyces cerevisiae strains with a defect in the ngd29 gene (e.g.DSM 7042/7338) and/or ngd62 gene (e.g. DSM 7160/7340) are transformedwith a recombinant DNA which contains the gene for GOD and, afterculture of the cells, the glucose oxidase is isolated from the cells orfrom the supernatant.

Finally the invention in addition concerns the use of a glucose oxidaseaccording to the invention in a diagnostic test.

For patent purposes the following were deposited under the BudapestTreaty at the "Deutsche Sammlung fur Mikroorganismen und ZellkulturenGmbH (DSM)", Mascheroder Weg 1 B, D-3300 Braunschweig. Subject to 37 CFR1.808(b), all restrictions imposed by the depositor on the availabilityto the public of the deposited material will be irrevocably removed upongranting of the patent.

    ______________________________________                                                           Deposit Deposit                                                               number  date                                               ______________________________________                                        1.  Plasmid YEpL         DSM 7038  07.04.1992                                 2.  Yeast mutant BMY3-9A (ngd29)                                                                       DSM 7042  08.04.1992                                 3.  Yeast mutant BMY3-9C (ngd29)                                                                       DSM 7193  24.07.1992                                 4.  Yeast mutant BMY12-20D (ngd62)                                                                     DSM 7160  09.07.1992                                 5.  Yeast mutant BMY8-12A (ngd62)                                                                      DSM 7157  09.07.1992                                 6.  Yeast mutant BMY13-7B (mnn9)                                                                       DSM 7158  09.07.1992                                 7.  Yeast mutant BMY13-1C (mnn9)                                                                       DSM 7159  09.07.1992                                 8.  Yeast mutant JM 1935 DSM 7156  09.07.1992                                 9.  Yeast mutant DBY 746 DSM 4316  14.12.1987                                 10. Yeast mutant N-BMY3-9A                                                                             DSM 7338  08.12.1992                                 11. Yeast mutant N-BMY13-1C                                                                            DSM 7339  08.12.1992                                 12. Yeast mutant N-BMY12-20D                                                                           DSM 7340  08.12.1992                                 13. Yeast mutant N-BMY3-9C                                                                             DSM 7341  08.12.1992                                 ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plasmid YEp/5C6b3.

FIG. 2 depicts the plasmid YEpL.

FIG. 3 depicts the plasmid YEp/GOD.

The following examples elucidate the invention further.

EXAMPLES

General Methods

Recombinant DNA Technique

Standard methods were used to manipulate DNA such as those described byManiatis, T. et al., in: Molecular cloning: A laboratory manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989). Themolecular biological reagents used were used according to themanufacturer's instructions.

Yeast Transformation

Saccharomyces cerevisiae strains were transformed according to themethod of Beggs, J. D. (Nature 275 (1978) 104-109; Ito, H. et al., J.Bacteriol. 153 (1983) 163-168 or Delorme, E. (Applied and EnvironmentalMicrobiology 55 (1989) 2242-2246). Fructose was used instead of glucoseas a C source.

Determination of Glucose Oxidase Activity

The determination of GOD activity was carried out at 25° C. in a volumeof 1 ml in 0.1 mol/l potassium phosphate buffer, pH 7.0 saturated withoxygen containing 0.18 mol/l glucose, 15 units/ml horse-radishperoxidase and 1.75 mmol/l ABTS® glucose reagent. The reaction wasstarted by addition of glucose oxidase (10 μl sample containing GODdiluted to 5-20 mU/ml) and the change in absorbance/min (ΔA/min) wasdetermined at 405 nm (ε₄₀₅ =36.8 [mmol⁻¹ ×1×cm⁻¹ ]). 1 unit (U) GODactivity is defined as the amount of enzyme which oxidizes 1 μmolglucose per min at 25° C. The specific activity of purified A. niger GODis ca. 230 U/mg protein under these test conditions.

Protein Determinations

The protein determination was carried out by the microbiuret method(Zamenhof, S. et al., Methods Enzymol. 3 (1957) 696-704) with bovineserum albumin as standard.

The protein concentration of purified GOD enzymes was calculated on thebasis of the optical density at 280 nm (1 OD₂₈₀ ≅1.5 mg/ml purifiedGOD).

Cell Lysis and Isolation of Crude Extract

The cells from 5 ml culture medium (ca. 0.1-0.2 g yeast, wet weight)were centrifuged down. The cell pellet was washed once with 10 mmol/lphosphate buffer, pH 7.0 and subsequently lysed with glass beads byhomogenization with a Whirlmix (Ciriacy, M., Mut. Res. 29 (1975)315-326). Afterwards the cells were resuspended/extracted in 2 ml 10mmol/l phosphate buffer, pH 7.0, the cell debris was removed bycentrifugation and the supernatant was processed further as a crudeextract.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Soluble samples (medium supernatants and cell lysates) were admixed with1/5 volumes 5×SDS sample buffer (1×SDS sample buffer: 50 mmol/lTris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol, 0.001%bromophenol blue) and incubated for 5 min at 95° C. Non-soluble proteinsof the cell debris fraction were extracted with 2 ml 1×SDS sample bufferand 6-8 mol/l urea, denatured by heating for 5 minutes to 95° C. andseparated from insoluble components by centrifugation. Afterwards theproteins were separated by SDS-PAGE (Laemmli, U.K., Nature 227 (1970)680-685) and stained with Coomassie Brilliant Blue® dye.

Example 1 Construction of Plasmids for the Secretion of A. niger GOD inS. cerevisiae Construction of the Yeast Expression Vector YEpL (StartingVector)

Plasmid YEpL is based on the α-glucosidase vector YEp/5C6b3 (Kopetzki etal., Yeast 5 (1989) 11-24; Kopetzki, et al., EP-A 0 323 838). The ca.2.3 kBp long EcoRI/PvuII fragment from the plasmid pBR322 (plasmidorigin, ampicillin resistance gene) serves to replicate the plasmid inE. coli. For replication in yeast the vector contains the ca. 2.2 kBplong EcoRI fragment from the 2 μm DNA of yeast (subcloned from the E.coli/yeast shuttle vector YEp24). In addition the vector contains theURA3 and LEU2d gene in order to select the plasmid in auxotrophic yeaststrains and an α-glucosidase expression cassette (GLUCPI gene). Itconsists of the α-glucosidase promoter, a polylinker (cloning site forthe genes to be expressed) and the α-glucosidase terminator. In additionthe MAL2-8cp gene is present the gene product of which, the MAL2-8cpprotein, activates the α-glucosidase promoter. The α-glucosidasepromoter is repressed in the presence of glucose. It derepresses afterconsumption of the glucose and achieves its maximum activity afterinduction with maltose.

1.1 Construction of the Plasmid YEp/KL6b3

The ca. 1.4 kBp long DNA sequence which is not required between theα-glucosidase terminator and the MAL2-8cp promoter was deleted from theplasmid YEp/5C6b3 (FIG. 1).

For this the plasmid YEp/5C6b3 was linearised with XhoI, the 5'overhanging ends were filled up with Klenow polymerase, the plasmid wasre-cleaved with MroI and the 8.7 kBp long MroI/XhoI (blunt) vectorfragment was isolated. In a second preparation the plasmid YEp/5C6b3 wasdisgested with the restriction endonucleases MroI and ScaI, the 2.5 kBplong MroI/ScaI fragment containing the α-glucosidase gene was isolatedand ligated with the 8.7 kBp long MroI/XhoI(blunt) vector fragment. Thedesired plasmid was identified by restriction mapping and designatedYEp/KL-6b3.

1.2 Construction of the Plasmid YEp/KL-6b3M

A MluI-linker (5'-GACGCGTC-3') was ligated into the SspI restrictionendonuclease cleavage site of the 5' non-translated region of theMAL2-8cp gene. Plasmid construction: YEp/KL-6b3M.

1.3 Construction of the Plasmid YEp/KL-6b3M-MCS

The structural gene of α-glucosidase was removed by the "polymerasechain reaction" (PCR) technique (Mullis, K. B. and Faloona, F. A.,Methods in Enzymol. 155 (1987) 335-350) and replaced by a DNA linker(multicloning site, MCS). ##STR1##

For this the GLUCPI promoter sequence from the plasmid YEp/KL-6b3M wasamplified by means of PCR using the primer pair (see SEQ ID NO. 1 andSEQ ID NO. 2) ##STR2## and the ca. 410 Bp long PCR product was isolatedby agarose gel electrophoresis.

The GLUCPI terminator sequence from the plasmid YEp/KL-6b3M wasamplified in a second PCR reaction using the primer pair (see SEQ ID NO.3 and SEQ ID NO.4) ##STR3## and the ca. 860 Bp long PCR product wasisolated by agarose gel electrophoresis.

Afterwards equimolar amounts (ca. 50 pg of each) of the isolated PCRfragments were combined in the PCR reaction mixture, incubated for 5 minat 95° C. to denature the ds-DNA, the reaction mixture was cooled to 60°C. to anneal the complementary single DNA strands containing MCS, thehybridization products were converted into ds-DNA using Taq polymeraseand amplified in a third PCR reaction using the primer pair (see SEQ IDNO. 1 and SEQ ID NO. 4) ##STR4##

Afterwards the ca. 1.27 kBp long PCR product was digested with therestriction endonucleases MroI and MluI, the ca. 0.92 kBp longMroI/MluI-GLUPI-promoter/MCS/GLUCPI terminator fragment was isolated byagarose gel electrophoresis and ligated into the ca. 8.55 kBp longMroI/MluI-YEp/KL-6b3M vector fragment. The desired plasmidYEp/KL-6b3M-MCS was identified by means of restriction mapping and theDNA regions synthesized by means of PCR were checked by DNA sequencing.

1.4 Construction of the Plasmid YEpL

In the following plasmid construction the LEU2d gene was inserted intothe plasmid YEp/KL-6b3M-MCS. For this the plasmid YEp/KL-6B3M-MCS wasdigested with CelII and SnaBI and the 8.4 kBp longCelII/ScaBI-YEp/KL-6b3M-MCS vector fragment was isolated. The LEU2d genewas isolated as a ca. 2.32 kBp long CelII/SnaBI fragment from theplasmid pADH040-2 (Erhart, E. and Hollenberg, C. P., J. Bacteriol. 156(1983) 625-635) and ligated with the 8.4 kBp longCelII/SnaBI-YEp/KL-6b3M-MCS vector fragment. The desired plasmidconstruction YEpL (DSM 7038) was identified by restriction mapping (FIG.2).

1.5 Construction of the Plasmid YEpL/GOD

The cloning of the glucose oxidase gene used (strain: NRRL-3, ATTC9029), subcloning in the pBluescript SK(+) vector, DNA sequencing anddeduction of the GOD protein sequence are described in the publicationof Kriechbaum, M. et al. (FEBS Lett. 255 (1989) 63-66). The GOD gene wascloned in 2 partial regions (SalI restriction fragments) intopBluescript SK(+).

The plasmid pSK/GOD-1.8 contains a ca. 1.8 kBp long SalI fragment whichcodes for the 5'-non-translated region and N-terminal region of the GODstructural gene up to the SalI cleavage site at Bp position 164 (Bpposition corresponds to the numbering by Kriechbaum, M. et al). Theplasmid pSK/GOD-2.0 contains a ca. 2.0 kBp long SalI fragment whichcodes for the remainder of the GOD structural gene from Bp position 165to 1853 as well as for the 3'-non-translated region downstream of theGOD structural gene.

The 5'- and 3'-non-translated region of the GOD gene was removed bymeans of the PCR technique, both ends of the GOD structural gene wereprovided with single restriction endonuclease cleavage sites (BglII andPvuII) and in addition a single SphI and NheI cleavage site wereintroduced into the C-terminal coding region of the GOD structural genewhile maintaining a DNA sequence coding for the native GOD protein.Subsequently the GOD structural gene was assembled from the two PCRfragments in a three-fragment ligation and inserted into the vectorYEpL. The following primer pair (see SEQ ID NO. 5 and SEQ ID NO. 6) wasused to amplify the N-terminal GOD structural gene and plasmidpSK/GOD-1.8 was used as template DNA. ##STR5##

The following primer pair (see SEQ ID NO. 7 and SEQ ID NO. 8) was usedto amplify the remaining GOD structural gene and plasmid pSK/GOD-2.0 wasused as template DNA. ##STR6##

The ca. 220 Bp long PCR product of the first reaction was re-cleavedwith BglII and SalI and the ca. 130 Bp long BglII/SalI fragment wasisolated. The ca. 2.05 kBp long PCR product of the second reaction wasdigested with SalI and PvuII and the ca. 1.7 kBp long DNA fragment wasisolated. Afterwards the PCR fragments were ligated into the ca. 10.7kBp long BglII/PvuII-YEpL vector fragment (three-fragment ligation). Thedesired plasmid YEpL/GOD (FIG. 3) was identified by restriction mappingand partially sequenced (cloning junctions).

1.6 Construction of the Plasmid YEpL/GOD-(His)4

The plasmid contains a modified GOD gene which codes for a GOD enzymevariant that has four additional histidine residues at the C-terminus.YEpL/GOD-(His)₄ was produced from the plasmid YEpL/GOD.

For this the plasmid YEpL/GOD was partially cleaved with SphI andcompletely cleaved with PvuII, the ca. 10.7 kBp long SphI/PvuII fragmentwas isolated and ligated with the following DNA linker prepared from twooligonucleotides (see SEQ ID NO. 9 and SEQ ID NO. 10) by hybridization.##STR7##

The desired plasmid YEpL/GOD-(His)4 was identified by colonyhybridization using radioactively-labelled primer 10 as the probe andfurther analyzed by restriction mapping and partial sequencing(C-terminal region of the GOD structural gene).

Example 2 Isolation of Yeast Host Strains with Defective N-glycosylation2.1 [³ H]-mannose Suicide Mutagenesis

Principle:

Mutagenesis (starting strain: X2180-1A, genotype: a SUC2 mal mel gal2CUP1; ATCC 26786)

Incubation with [³ H]-mannose

Accumulation of hyperglycosylation-defective mutants by storing thecells at -80° C. until the survival rate of the cells decreases to 10²-10³ (2-4 months)

A yeast strain such as X2180-1A (ATCC 26786) is cultured in YEPD medium(2% Bactopeptone, 1% yeast extract, Difco, and 4% glucose), harvested inthe logarithmic growth phase (ca. 5×10⁸ of these cells), washed with 0.1mol/l sodium phosphate, pH 7 and resuspended in 1.5 ml 0.1 mol/l sodiumphosphate, pH 7. The cells are mutagenized by the addition of 0.1 mlethyl methanesulfonate for 1 hour at 25° C. 0.2 ml of the cells treatedin this way is incubated for 10 minutes with 10 ml sodium thiosulfate(5% w/v), washed 3× with 0.1 mol/l sodium phosphate, pH 7.0 andresuspended in YEPD medium (2% Bactopeptone, 1% yeast extract, Difco and4% glucose).

The cells are incubated at 28° C. while shaking until an OD of 0.6 at578 nm is achieved. 10⁶ cells are washed with YEP medium (2%Bactopeptone, 1% yeast extract, Difco) and resuspended in 0.1 ml YEPcontaining 0.1% glucose. 2 mCi [³ H]-mannose (specific activity 18.5Ci/mmol) is added and the culture is incubated for 60 minutes at 28° C.The cells are centrifuged, washed with water and resuspended in YEPDwhich contains 25% glycerol and stored at -70° C. for the radioactivityto take effect. After ca. 45-50 days when the survival rate of the cellshas dropped to 1.5-0.2%, aliquots of the cells are plated on YEP agarplates containing 2% mannose and incubated at 30° C.

2.2 Isolation of Mutants with Reduced N-glycosylation

Mutants with a defect in protein glycosylation are firstly selected fortheir ability not to incorporate [³ H]-mannose and to incorporate [³⁵S]-methionine. For this the cells are allowed to grow on YEPD agarplates, the yeast colonies are replicated on 2 Rotband filters(Schleicher & Schell, Dassel, Germany) and the filters are incubatedagain for 6 hours on YEPD plates. One filter is then incubated in asolution of YEPD (an amount which is just sufficient to wet the filter)which contains 0.01 mCi/ml [³⁵ S]-methionine. The other filter isimpregnated with YEP containing 0.2 mCi/ml [³ H]-mannose and incubatedfor 30 minutes. The cells/colonies are immobilized on the filter with 5%trichloroacetic acid, washed with water and acetone and analyzed byautoradiography.

2.3 Characterization of Positive Clones by Native Gel Electrophoresis ofExternal Invertase

The SUC2 gene from S. cerevisiae codes for 2 different regulated andcompartmented invertase forms, i) a glycosylated invertase which ismainly secreted into the periplasm and ii) an intracellular slightlytruncated non-glycosylated form (Carlson, M. et al., Mol. Cell. Biol. 3(1983) 439-447). Invertase contains 14 potential N-glycosylation sitesof which 9-10 are glycosylated on average in the secreted form perinvertase subunit. External wild-type invertase migrates as a diffuseband in native gels due to non-uniform outer chain glycosylation. Incontrast the cytoplasmic non-glycosylated form yields a sharp band afteractivity staining. A change in N-glycosylation can thus be crudelyanalyzed by means of the migration rate and band sharpness of externalinvertase in native gels.

The yeast strains (X2180-1A wild-type strain and positive clones) werecultured overnight in 5 ml YEPS medium (1% yeast extract, 2%Bactopeptone, Difco and 2% sucrose), the cells were harvested in thelate logarithmic growth phase, washed once with 20 mmol/l sodium azideand lysed with glass beads by homogenization in a Whirlmix. Thepreparation of cell lysate, the native gel electrophoresis and activitystaining of invertase with sucrose and 2,3,4-trinitrophenyltetrazoliumchloride as substrate/glucose reagent were carried out according to themethod of Ballou C. E. (Methods Enzymol. 185 (1990) 440-470).

The positive clones may be divided into 4 classes on the basis of theinvertase activity staining:

1. Mutants with wild-type invertase mobility.

2. Mutants that synthesize neither non-glycosylated nor glycosylatedinvertase.

3. Mutants with defects in outer chain glycosylation (distinctoligomeric band pattern of 3-4 bands).

4. Mutants which lead to a substantial under-glycosylation of invertase(larger mobility than wild-type invertase).

Results

Mutant strains of class 4, designated ngd29 (DSM 7042/7338) and ngd62(DSM 7160/7340) in the following (ngd stands for"N-glycosylation-defective"), synthesize in comparison to the startingstrain X2180-1A a uniformly glycosylated dimeric external invertase(sharp bands and increased migration rate in native gels after activitystaining). The ngd mutant strains were osmotically stable, could becultured at 30° C. and did not aggregate during culture.

Example 3 Construction of Glycosylation-defective Yeast Host Strains forthe Expression of Homologous and Heterologous Proteins

In order to introduce one or several auxotrophies which can becomplemented by transformation, the ngd mutants were crossed withsuitable laboratory strains according to the method described by F.Sherman et al. (Methods in Yeast Genetics: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., (1981)) and diploidstrains were isolated by micromanipulation. Subsequently the diploidstrains were sporulated and segregants with suitable auxotrophies (e.g.ura3, leu2) and ngd mutation were isolated.

For this the ngd29 mutant was incubated together with the strain DBY746(MATα ura3-52 leu2-3,-112 trp1-289α his3-α1; [DSM 4316] equivalent toATCC 44733) and the ngd62 mutant was incubated with the JM1935 strain(MATα ura3 leu2 his4, DMS 7156) for 6 hours at 30° C. in YEPD (1% yeastextract, 2% Bactopeptone, Difco, and 2% glucose). Subsequently zygoteswere isolated with the aid of a micromanipulator (model according to deFonbrune from the Bachhofer Company, Reutlingen, Germany) and grownovernight in 5 ml YEPD. The cells were briefly centrifuged, the mediumwas decanted until about 0.2 ml remains and the cell pellet wasresuspended in the residual medium. This cell suspension was plated on apotassium acetate plate (1% potassium acetate, 1.5% agar). After ca. 5days the asci obtained in this way were resuspended in 0.5 ml sterilewater using an inoculating loop, 10 μl of a β-glucuronidase/arylsulfatase mixture (Boehringer Mannheim) was added and it was incubatedfor 10 min at room temperature. Subsequently 10 ml water was added, theascus suspension was centrifuged and the supernatant was decanted. Thespores of several asci were isolated under a micromanipulator andincubatedon YEPD plates (YEPD containing 1.5% agar) for 3 days at 30° C.A replica plate was prepared from germinated spores, the colonies werepressed onto synthetic minimal media (0.67% yeast nitrogen base withoutamino acids, Difco; 2% glucose; 1.5% agar plus additives: 20 mg/l Trp,His, Arg, Met; 30 mg/l Leu, Ile, Lys; 50 mg/l Phe; 100 mg/l Glu, Asp;400 mg/l Val, Thr, Ser as well as 20 mg/l adenine and uracil; one ofthese additives was omitted in each of the individual minimal media) andincubated for 3 days at 30° C. Segregants with the ngd29 phenotype whichhave auxotrophies for uracil and leucine were analyzed/isolated asdescribed in example 2.2. The ngd29 phenotype (as well as the ngd62phenotype) segregated 2:2 in all examined tetrads, which indicates asingle mutation in a single nuclear locus.

Strains BMY3-9A and N-BMY3-9A (MATα leu2-3,-112 ura3-52 his3-Δ1 ngd29;DSM 7042 and DSM 7338) and BMY3-9C and N-BMY3-9C (MATα leu2-3, -112,ura3-52 ngd29; DSM 7193 and DSM 7341) were obtained from the crossDBY746 x ngd29 described here.

Strains BMY12-20D and N-BMY12-20D (MATα leu2 ura3 his4 ngd62; DSM 7160and DSM 7340) were obtained in an analogous manner from the cross JM1935x ngd62.

Example 4 Comparison of the Expression/Secretion of Native A. niger GODand the GOD (His)₄ Variant in Wild-type and Glycosylation-defectiveYeast Host Strains

The GOD from A. niger is a naturally secreted glycosylated dimericenzyme. 8 potential N-glycosylation sites (sequons) and 3 cysteineresidues two of which form a disulfide bridge are present per subunit.GOD expressed in S. cerevisiae wild-type strains is secreted into themedium and is very heterogeneous with regard to molecular weight due toa non-uniform outer chain glycosylation (hyperglycosylation) (Frederick,K. R. et al., J. Biol. Chem. 265 (1990) 3793-3802; De Baetselier, A. etal., Biotechnology 9 (1991) 559-561; Whittington, H. et al., Curr.Genet. 18 (1990) 531-536). The processed (cleavage of a 22 amino acidlong signal sequence) A. niger GOD protein consists of 583 amino acidswith a potential molecular weight of 63 273 Da (Frederick, K. R. et al.,J. Biol. Chem. 265 (1990) 3793-3802).

The plasmids YEpL/GOD (example 1.5) and YEp/GOD-(His)₄ (example 1.6)were transformed into the wild-type strain JM1935 (MATα leu2 ura3 his4MAL4) DSM 7156 N-BMY3-9A or BMY3-9A (see example 3) and thetransformants were selected on minimal medium agar plates containing1.5% agarose, 0.67% YNB (yeast nitrogen base, salt-vitamin mixture,Difco) 0.5% CAA (casamino acids, protein hydrolysate, Difco) and 2%fructose as a C-source (uracil selection).

4.1 Culture of the GOD Transformants

In order to amplify the plasmid copy number (selection for the plasmidcoded LEU2d allele; Beggs, J. D., Nature 275 (1978) 104-109; Erhart, E.and Hollenberg, C. P. J., Bacteriol. 156 (1983) 625-635) thetransformants were streaked on minimal medium plates without leucine(1.5% agarose, 0.67% YNB, Difco, 60 mg/l adenine and 2% fructose).

Precultures were carried out in leucine selective medium containing0.67% YNB and 4% fructose in shaking flasks at 30° C. for 48 hours andused to inoculate expression cultures (inoculum: 1-2%). The main culture(1 l shaking culture) was incubated at 30° C. in complete mediumcontaining 2% yeast extract, 4% Bactopeptone, Difco, 0.1 mol/l phosphatebuffer, pH 7.0, 1% fructose and 6% maltose for 3-4 days while shaking.Samples were taken after 48 and 72 hours and the cell growth(determination of the optical density at 600 nm, OD₆₀₀), the GODactivity secreted into the medium and residual GOD activity in the cellswas determined in the crude extract after cell lysis.

    ______________________________________                                        Expression/secretion analysis of GOD in the wild-type                         strain DSM 7156 and the glycosylation-defective host                          strains DSM 7042 or DSM 7338                                                  Plasmid: YEpL/GOD                                                                     GOD activity (U/ml) / Optical density (OD.sub.600)                            Time (hours)                                                                      48                 72                                             DSM 7156    U/ml / OD.sub.600  U/ml / OD.sub.600                              ______________________________________                                        extracellular                                                                              8     13            12   17                                      intracellular                                                                              4                    6                                           total       12                   18                                           % secreted  66                   66                                           ______________________________________                                        Plasmid YEpL/GOD                                                                      GOD activity (U/ml) / Optical density (OD.sub.600)                            Time (hours)                                                                      48                 72                                             DSM 7042/7338                                                                             U/ml / OD.sub.600  U/ml / OD.sub.600                              ______________________________________                                        extracellular                                                                             11     9             18   14                                      intracellular                                                                              1                    2                                           total       12                   20                                           % secreted  87                   90                                           ______________________________________                                        Expression/secretion analysis of GOD-(His).sub.4 in the wild-                 type strain DSM 7156 and the glycosylation-defective                          host strains DSM 7042/7338                                                    Plasmid: YEpL/GOD-(His).sub.4                                                         GOD activity (U/ml) / Optical density (OD.sub.600)                            Time (hours)                                                                      48                 72                                             DSM 7156    U/ml / OD.sub.600  U/ml / OD.sub.600                              ______________________________________                                        extracellular                                                                              8     14             9   14                                      intracellular                                                                              5                    6                                           total       13                   16                                           % secreted  62                   58                                           ______________________________________                                        Plasmid: YEpL/GOD                                                                     GOD activity (U/ml) / Optical density (OD.sub.600)                            Time (hours)                                                                      48                 72                                             DSM 7042/7338                                                                             U/ml / OD.sub.600  U/ml / OD.sub.600                              ______________________________________                                        extracellular                                                                             12     10            17   13                                      intracellular                                                                              1                    1                                           total       13                   18                                           % secreted  88                   93                                           ______________________________________                                    

Result

No significant differences were found between the GOD and GOD-(His)₄variant with regard to expression and secretion.

4.2 SDS-PAGE of Secreted GOD

The GOD-(His)₄ enzyme expressed (secreted into the medium) in theglycosylation-defective host strains DSM 7042/7338 (ngd29) and DSM7160/7340 (ngd62) together with the enzyme expressed (secreted) in thewild-type strain DSM 7156 and purified GOD from A. niger (BoehringerMannhein, GFR) were further characterized by SDS-PAGE and subsequentprotein staining. The medium supernatants from the wild-type straincontaining GOD were concentrated 10-fold by TCA precipitation beforeelectrophoresis. Carbohydrate-free GOD-(His)₄ enzyme was preparedenzymatically using N-glycosidase F and used as a standard for size.

Enzymatic Deglycosylation with N-glycosidase F

The deglycosylation was carried out according to the method published byHaselbeck, A. and Hosel, W. (Topics in Biochemistry 8 (1988) 1-4). 0.1ml medium supernatant containing GOD-(His)₄ was precipitated withtrichloroacetic acid (final concentration: 10%), the precipitatedproteins were centrifuged, the protein pellet was washed with 70%ethanol, dried in a vacuum, taken up in 10 μl 20 mmol/l potassiumphosphate buffer, pH 7.2 containing 1% SDS and heated for 3 min. to 95°C. After cooling to room temperature the sample was diluted to 0.1 mlwith 20 mmol/l potassium phosphate buffer, pH 7.2, octylglucoside (finalconcentration: 0.5%) and 5 units N-glycosidase F, incubated for 1-12hours at 37° C. and subsequently 25 μl 5×SDS buffer (see above) wasadded.

Result

The GOD enzymes (GOD and GOD-(His₄)) expressed in theglycosylation-defective ngd mutant strains are visible in SDS-PAGE gelsafter protein staining as dominant uniform bands with a molecular weightof ca. 80 kDa. This experiment shows the absence of outer chainglycosylation in the GOD enzymes and indicates a uniform core-likeglycosylation. With regard to glycosylation the ngd mutant strains havea mnn9-like phenotype. In contrast the GOD enzymes expressed inwild-type strains are only recognizable as very diffuse bands whichcover a molecular weight range of ca. 80-200 kDa.

Example 5 Characterization of the N-glycosylation-defective ngd Mutantson the Basis of Growth on YEPD Agar Plates Containing Orthovanadate orHygromycin B

Glycosylation-defective mutants such as e.g. mnn8, mnn9 and mnn10 showan increased resistance to orthovanadate and an increased sensitivity tothe antibiotic hygromycin B. The resistance/sensitivity phenotypeenables a differentiation/classification of N-glycosylation-defectivemutants (Ballou, L. et al., Proc. Natl. Acad. Sci. 88 (1991) 3209-3212).

The strains to be examined were cultured overnight in YEPD medium (5 mlroller culture) and the strains/cultures were adjusted to an opticaldensity (OD₆₀₀) of exactly 0.05 with YEPD medium. Afterwards 20 μl ofeach cell suspension was spotted on YEPD agar plates containing 2-15mmol/l sodium orthovanadate or 10-200 μg/ml hygromycin B. The growth ofthe cell spots was evaluated after 2 days incubation at 30° C. (seetable).

    __________________________________________________________________________    Growth phenotype of yeast cells on YEPD agar Plates                           containing sodium orthovanadate or hygromycin B                                                           Hygromycin                                                    Orthovanadate resistance                                                                      resistance                                                    mmol/l          (μg/ml)                                        Strain      2 3 4 5 6 7 10                                                                              15                                                                              10                                                                              50                                                                              100                                                                              200                                        __________________________________________________________________________    DBY 746 (wild-type)                                                                       + + + + - - - - + + -  -                                          X2180-1A (wild-type                                                                       + + + + - - - - + + -  -                                          LB347-1C (mnn9).sup.1)                                                                    + + + + + + + + - - -  -                                          BMY3-9A (ngd29)                                                                           + + + + + - - - + ±                                                                            -  -                                          BMY12-20D (ngd62)                                                                         + + + ±                                                                            - - - - + ±                                                                            -  -                                          N-BMY3-9A (ngd29)                                                                         + + + + + - - - + ±                                                                            -  -                                          N-BMY12-20D (ngd62)                                                                       + + + ±                                                                            - - - - + ±                                                                            -  -                                          __________________________________________________________________________     + growth                                                                      ± very slow growth                                                         - no growth                                                                   .sup.1) J. Biol. Chem. 259 (1984) 3805-3811                              

Result

There are differences between the ngd mutants with regard to resistancepattern which differs from that of the mnn9 mutant and wild-typestrains.

Example 6 Characterization/Identification of ngd Mutants (Allelism Test)

An allelism test serves to identify (differentiate between) genes andgene defects (mutations). With this technique it is possible to analyzewhether 2 mutants are allelic (have a mutation in the same gene). Thengd mutants were examined for allelism among each other and to the mnn9mutant.

The allelism tests were carried out by means of genetic standardtechniques (see: Sherman, F.; Fink, G. R.; Hicks, J. B., Methods inYeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., (1981); Guthrie, C. and Fink, G. R. (eds.), Guideto Yeast Genetics and Molecular Biology. Methods Enzymol. 194 (1991)).

Principle:

Two haploid mutant strains to be analyzed of different pairing type withauxotrophy requirements that complement each other are crossed and thediploid strains are selected on plates with minimal medium. Thediploidism of the isolated strains is confirmed by the presence of DNAsequences specific for the a and α pairing type using PCR analysis inaccordance with the method of Huxley, C. et al. (Trends Genet. 6 (1990)236).

Two mutants are allelic, i.e. have a mutation in the same gene, when themutations do not complement each other in the diploid cell.

Two mutants are not allelic, i.e. have a mutation in two differentgenes, when the mutations complement each other in the diploid cell anda wild-type phenotype results.

Strains used:

    __________________________________________________________________________    BMY3-9C (MATα leu2-3,-112 ura3-52 ngd29)                                                              DSM 7193                                        BMY8-12A (MATa trp1-289.sup.a his3-Δ1 ngd62)                                                          DSM 7157                                        BMY13-1C (MATα ura3-52 leu2-3,-112 his3-Δ1 mnn9)                                                DSM 7159                                        BMY13-7B (MATa leu2-3,-112 his3-Δ1 mnn9)                                                              DSM 7158                                        BMY12-20D (MATα leu2 ura3 his4 ngd62)                                                                 DSM 7160                                        N-BMY3-9C (MATα leu2-3,-112 ura3-52 ngd29)                                                            DSM 7341                                        N-BMY13-1C (MATα ura3-52 leu2-3,-112 his3-Δ1                                                    DSM 7339                                        BMY12-20D(MATα leu2 ura3 his4 ngd62)                                                                  DSM 7340                                        Crossing partners                                                                             Phenotype of                                                                          Selection                                                                           Phenotype of                                    MATα MATa the haploids                                                                          diploids                                                                            the diploids                                    __________________________________________________________________________    BMY3-9C × BMY8-12A                                                                      ngd29xngd62                                                                           his leu                                                                             wild-type                                       BMY3-9C × BMY13-7B                                                                      ngd29xmnn9                                                                            his ura                                                                             wild-type                                       BMY13-lC                                                                              × BMY8-12A                                                                      mnn9xngd62                                                                            trp ura                                                                             wild-type                                       BMY12-20D                                                                             × BMY13-7B                                                                      ngd62xmnn9                                                                            his   wild-type                                       N-BMY3-9C                                                                             × BMY8-12A                                                                      ngd29xngd62                                                                           his leu                                                                             wild-type                                       N-BMY3-9C                                                                             × BMY13-7B                                                                      ngd29xmnn9                                                                            his ura                                                                             wild-type                                       N-BMY13-lC                                                                            × BMY8-12A                                                                      mnn9xngd62                                                                            trp ura                                                                             wild-type                                       N-BMY12-20D                                                                           × BMY13-7B                                                                      ngd62xmnn9                                                                            his   wild-type                                       __________________________________________________________________________

SC=synthetic complete medium (0.67% yeast nitrogen base without aminoacids, Difco; 2% glucose; 1.5% agar plus additives: 20 mg/l Trp, His,Arg, Met; 30 mg/l Leu, Ile, Lys; 50 mg/l Phe; 100 mg/l Glu, Asp; 400mg/l Val, Thr, Ser as well as 20 mg/l adenine and uracil; the aminoacids Ura, His and Trp were omitted in the individual minimal media asstated in the table in the column headed "Selection of diploids".

Result:

The mutants ngd29 and ngd62 differ from one another and are differentfrom mnn9 (non-allelic).

Example 7 Isolation of GOD and GOD-(His)₄ from Wild-type andHyperglycosylation-defective Yeast Strains 7.1 Isolation of GOD-(His)₄by means of Metal Chelate Chromatography

The GOD variant GOD-(His)₄ was isolated using this isolation method fromthe culture filtrate of BMY3-9A/GOD-(His)₄ cells andBMY12-20D/GOD-(His)₄ cells (hyperglycosylation-defective host strains).

The culture filtrate was titrated to pH 7.5 with sodium hydroxidesolution and applied to a NTA column equilibrated with 10 mmol/lpotassium phosphate buffer, pH 7.5 (column volume 25 ml; NTA gel fromthe Diagen Company, Dusseldorf; Hochuli, E. et al., J. Chromatography411 (1987) 177-184; Hochuli, E. et al., Biotechnology 6 (1988)1321-1325). The column was rewashed with 5-10 column volumes 1 mol/lsodium chloride in 10 mmol/l potassium phosphate buffer, pH 7.5, andwith 5-10 column volumes 10 mmol/l potassium phosphate buffer, pH 7.5.Afterwards the GOD-(His)₄ enzyme was eluted with 0.1 mol/l imidazole inequilibration buffer, pH 7.5 and the fractions containing GOD-(His)₄(yellow) were dialysed against 10 mmol/l potassium phosphate buffer, pH7.5.

7.2 Isolation of GOD and GOD Variants by Ion Exchange Chromatography onQ-Sepharose ff After Previous Concentration and Dialysis

Native GOD and hyperglycosylated GOD were purified according to thismethod.

33 g solid ammonium sulfate (AS saturation concentration 55%) was addedto 100 ml sterile-filtered culture filtrate while stirring slowly, theprecipitated proteins were centrifuged down after 1-2 hours incubationat room temperature, dissolved in 25 ml 25 mmol/l potassium phosphatebuffer, pH 7.5 and dialysed against the same buffer (4×10 l, 24 hours,4° C.).

Subsequently the dialysate was applied to a Q-Sepharose ff column(column volume 12 ml) equilibrated with 25 mmol/l potassium phosphatebuffer, pH 7.5 and rewashed with 5-10 column volumes equilibrationbuffer. The bound GOD enzymes were eluted by a gradient of 0-1 mol/l KClin equilibration buffer (ca. 10 column volumes) and the fractionscontaining GOD (yellow) were pooled.

Example 8 Biochemical Characterization of the Isolated GOD Enzymes 8.1Determination of the Specific GOD Activity

The determination of GOD activity is carried out as described in the"general methods" section.

    ______________________________________                                        Specific activity of GOD and GOD-(His).sub.4 expressed in                     A. niger, S. cerevisiae (wild-type) and S. cerevisiae                         (hyperglycosylation-defective mutants)                                                  Organism/  spec. activity                                                                            spec. activity                               Enzyme    glycosylation                                                                            (U/mg protein)                                                                            (U/mg enzyme)                                ______________________________________                                        GOD       (A. niger) 225         195                                          GOD       (WT)       230          69                                          GOD       (ngd29)    228         196                                          GOD       (ngd62)    213         220                                          GOD-(His).sub.4                                                                         (WT)       220          68                                          GOD-(His).sub.4                                                                         (ngd29)    223         200                                          GOD-(His).sub.4                                                                         (ngd62)    230         225                                          ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  WT, S. cerevisiae wildtype                                                    ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant                 ngd62, S. cerevisiae hyperglycosylationdefective ngd62 mutant            

8.2 Determination of Molecular Weight by SDS Polyacrylamide GelElectrophoresis (SDS-PAGE)

The purified GOD enzymes were admixed with 1/5 volumes 5×SDS samplebuffer (1×SDS sample buffer: 50 mmol/l Tris-HCl, pH 6.8, 1% SDS, 1%mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and incubatedfor 5 min at 95° C. Afterwards the proteins were separated by SDS-PAGE(Laemmli, U. K., Nature 227 (1970) 680-685) and stained with CoomassieBrilliant BlueR dye.

    ______________________________________                                        Molecular weight/subunit after SDS-PAGE of GOD and GOD-                       (His).sub.4 expressed in A. niger, S. cerevisiae (wild-type)                  and S. cerevisiae hyperglycosylation-defective mutants.                                   Organism/   Molecular weight/subunit                              Enzyme      glycosylation                                                                             (kDa)                                                 ______________________________________                                        GOD         (A. niger)  ca. 80                                                GOD         (WT)        80-140                                                GOD         (ngd29)     ca. 80                                                GOD         (ngd62)     ca. 80                                                GOD-(His).sub.4                                                                           (WT)        80-140                                                GOD-(His).sub.4                                                                           (ngd29)     ca. 80                                                GOD-(His).sub.4                                                                           (ngd62)     ca. 80                                                ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  WT, S. cerevisiae wildtype                                                    ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant                 ngd62, S. cerevisiae hyperglycosylationdefective ngd62 mutant            

8.3 Determination of the Portion of Carbohydrate (Anthrone Reaction)

The carbohydrate portion of the GOD enzymes from different organisms andyeast strains was determined following the method of Ashwell, G.(Methods Enzymol. 3 (1957) 84).

For this 0.5 ml purified GOD enzyme (concentration 20-100 U/ml in H₂ O)was mixed with 5 ml anthrone reagent, the solution was incubated for 5minutes at 25° C. and afterwards heated for 15 minutes in a boilingwater bath. After the sample had been cooled to 25° C. the absorbancewas determined at 630 nm against a reagent blank. The portion ofcarbohydrate in the GOD sample was determined by means of a mannosecalibration curve with mannose standard solutions of 5, 25, 75 and 100μg/ml mannose which was set up at the same time.

Preparation of the Anthrone Reagent:

66 ml concentrated sulfuric acid is carefully diluted with 34 ml water.After cooling to 80° C., 50 mg anthrone and 1 g thiourea are dissolvedin the sulfuric acid. The anthrone reagent can be stored for two weeksat 4° C.

    ______________________________________                                        Carbohydrate Portion of GOD and GOD-(His).sub.4 expressed in                  A. niger, S. cerevisiae (wild-type) and S. cerevisiae                         (hyperglycosylation-defective mutants)                                                    Organism/   Carbohydrate portion (%)                              Enzyme      glycosylation                                                                             (relative to protein)                                 ______________________________________                                        GOD         (A. niger)  13                                                    GOD         (WT)        71                                                    GOD         (ngd29)     12.5                                                  GOD         (ngd62)     13                                                    GOD-(His).sub.4                                                                           (WT)        65                                                    GOD-(His).sub.4                                                                           (ngd29)     11                                                    GOD-(His).sub.4                                                                           (ngd62)     12                                                    ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  WT, S. cerevisiae wildtype                                                    ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant                 ngd62, S. cerevisiae hyperglycosylationdefective ngd62 mutant            

8.4 Determination of the Km Value

The Km value of the various GOD enzymes was determined according to theinstructions of Michal, G., Methods of Enzymatic Analysis, Vol. 1,Bergmeyer, H. U. (ed.) "Verlag Chemie Weinheim", Academic Press, NewYork and London, pp. 144-156 (1974).

    ______________________________________                                        Km value of GOD and GOD-(His).sub.4 expressed in A. niger,                    S. cerevisiae (wild-type) and S. cerevisiae (ngd29                            mutant                                                                                     Organism/                                                        Enzyme       glycosylation                                                                            Km [mol × 1.sup.-1 ]                            ______________________________________                                        GOD          (A. niger) 0.03                                                  GOD          (WT)       0.03                                                  GOD          (ngd29)    0.03                                                  GOD-(His).sub.4                                                                            (WT)       0.03                                                  GOD-(His).sub.4                                                                            (ngd29)    0.03                                                  ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  WT, S. cerevisiae wildtype                                                    ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant            

8.6 Determination of the Thermostability of GOD and GOD-(His)₄ Expressedin A. niger, S. cerevisiae (Wild-type) and S. cerevisiae (ngd29 Mutant)

The thermostability of the various GOD enzymes was determined bydifferential scanning calorimetry (DSC). For this the denaturation point(Tm) of the GOD enzymes was determined in a defined solvent (H₂ O), at adefined GOD protein concentration (20 mg/ml) and defined heating rate(10° C./min) (see Table).

    ______________________________________                                        Tm value (from DSC spectra) of GOD and GOD-(His).sub.4                        secreted in A. niger and S. cerevisiae (ngd29 mutant)                                  Organism/  differential scanning calorimetry                         Enzyme   glycosylation                                                                            Tm value (°C.)                                     ______________________________________                                        GOD      (A. niger) 68.5                                                      GOD      (ngd29)    74.7                                                      GOD-(His).sub.4                                                                        (ngd29)    75.8                                                      ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant            

Residual GOD Activity After a Thermal Stress at 55° C. of GOD andGOD-(His)₄ Expressed in A. niger and S. cerevisiae (ngd29 Mutant)

In order to determine the thermostability, the various GOD enzymes wereincubated at 55° C. at a concentration of 25 U/ml in 0.2 mol/l sodiumphosphate buffer, pH 7.5 and after 2 hours the residual GOD activity wasdetermined as described in the general methods section.

    ______________________________________                                        Residual GOD activity after a thermal stress of GOD and                       GOD-(His).sub.4 expressed in A. niger, S. cerevisiae (wild-                   type) and S. cerevisiae (ngd29 mutant)                                                    Organism/  Residual activity in %                                 Enzyme      glycosylation                                                                            after 2 h stress at 55° C.                      ______________________________________                                        GOD         (A. niger) 5                                                      GOD         (WT)       40                                                     GOD         (ngd29)    40                                                     GOD-(His).sub.4                                                                           (ngd29)    41                                                     ______________________________________                                         A. niger, GOD from A. niger, purity II (Boehringer Mannheim)                  WT, S. cerevisiae wildtype                                                    ngd29, S. cerevisiae hyperglycosylationdefective ngd29 mutant            

8.7 Determination of the pH Stability of GOD and GOD-(His)₄ Expressed inA. niger and S. cerevisiae (ngd29 Mutant)

The pH stability of the various GOD enzymes is determined by means ofDSC. For this the denaturation point (Tm) of the GOD enzymes wasdetermined in relation to the pH value in a defined solvent (100 mmol/lcitrate buffer for pH values of 3.5-6.5 and 100 mmol/l borate buffer forpH values of 7.5-9.5) at a defined GOD protein concentration (25 mg/ml)and a defined heating rate (10° C./minute).

Result:

GOD and GOD-(His)₄ isolated from the hyperglycosylation-defective ngd29mutant behaves like native Aspergillus niger GOD with respect to pHstability.

Publications:

Bekkers, A. C. A. P. A.; Franken, P. A. F.; Van den Bergh, C. J.;Verbakel, J. M. A.; Verheij, H. M.; De Haas, G. H.: The use of geneticengineering to obtain efficient production of porcine pancreaticphospholipase A2 by Saccharomyces cerevisiae. Biochim. Biophys. Acta1089, 345-351 (1991).

Ballou, L.; Cohen, R. E.; Ballou, C. E.: Saccharomyces cerevisiaemutants that make mannoproteins with a truncated carbohydrate outerchain. J. Biol. Chem. 255, 5986-5991 (1980).

Ballou, C. E.: Yeast cell wall and cell surface. In: Strathern, J. N.;Jones, E. W.; Broach, J. R. (eds.), The Molecular Biology of the YeastSaccharomyces, Metabolism and Gene Expression, Cold Spring HarborLaboratory, New York, pp. 335-360 (1982).

Ballou, L.; Alvarado, E.; Tsai, P.; Dell, A; Ballou, C. E.: Proteinglycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class.J. Biol. Chem. 264, 11857-11864 (1989).

Ballou, C. E.: Isolation characterization, and properties ofSaccharomyces cerevisiae mmn mutants with nonconditional proteinglycosylation defects. Methods Enzymol. 185, 440-470 (1990).

Ballou, L.; Hitzeman, R. A.; Lewis, M. S.; Ballou, C. S.:Vanadate-resistant yeast mutants are defective in protein glycosylation.Proc. Natl. Acad. Sci. 88, 3209-3212 (1991).

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATTTCTCCTTATTGCGCGCTT21                                                       (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TCTATTCAGCTGTCGACATAGATCTTATGTAATTTAGTTACGCTTGAC48                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      AGATCTATGTCGACAGCTGAATAGATAAAATTAGTGCGGACTTTTTTTTA50                          (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GTCATTTGTAAAGTAAAATTCCAA24                                                    (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GCCCGGTACCAGATCTATGCAGACTCTCCTTGTGAGCT38                                      (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      TCTAGAACTAGTGGATCCCCC21                                                       (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GCCGGCGAACGTGGCGAGAA20                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      ATATATCAGCTGTCACTGCATGCTAGCATAATCTTCCAAGATAGC45                               (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      CAGCACCACCACCACTGACAG21                                                       (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     CTGTCAGTGGTGGTGGTGCTGCATG25                                                   __________________________________________________________________________

We claim:
 1. Recombinant glucose oxidase obtainable from A. niger havinga molecular weight of about 68-80 kDa, a specific activity of about 200U/mg enzyme plus carbohydrate, a carbohydrate portion of about 12% and aresidual activity of at least 30% after incubation for two hours at atemperature of 55° C.
 2. A method of producing recombinant glucoseoxidase comprising transforming N-glycosylation deficient yeast cellsselected from the group consisting of DSM 7042, DSM 7338, DSM 7160 andDSM 7340 with a nucleic acid molecule from A. niger which encodesglucose oxidase and culturing said transformed yeast cells underconditions which allow for the production of glucose oxidase.